1. Field of the Invention
The invention relates to methods and apparatus for performing biological reactions on a substrate surface. More specifically, the invention relates to methods and apparatus for performing thermally controlled biological reactions on a substrate surface having one or more arrays of biologically reactive sites attached thereon. In particular, the invention provides a reusable and thermally controllable reaction apparatus having one or more biologically inert reaction chambers into which biologically reactive sample fluid mixtures are introduced for reaction on a substrate surface having one or more arrays of biologically reactive sites attached thereon.
2. Description of the Prior Art
As research into gene expression and nucleic acid sequencing has progressed in recent years, the need has arisen for high-capacity assaying methods and equipment. Much of the progress in the fields of nucleic acid sequencing and gene expression has resulted from the use of nucleic acid hybridization techniques and antigen/antibody binding techniques, respectively. Assays utilizing specific binding pairs such as complementary nucleic acids including DNA/DNA, DNA/RNA, and RNA/RNA hybrids or antigen/antibody are widely used in the art. The art also discloses various techniques for nucleic acid sequencing based on complementary binding and differential hybridization. Techniques for manufacturing and utilizing microfluidic apparatus for conducting such thermally controlled biological reactions are also well known.
Recent technology utilizes the binding of molecules contained within a biologically reactive sample fluid, hereinafter referred to as target molecules, onto molecules contained within biologically reactive sites, hereinafter referred to as probe molecules. The primary enabler of this technology is a apparatus commonly referred to as a biochip, which comprises one or more 2-dimensional microscopic arrays of biologically reactive sites immobilized on the surface of a substrate. A biologically reactive site is created by dispensing a small volume of biologically reactive fluid onto a discrete location on the surface of a substrate, also commonly referred to as spotting. To enhance immobilization of probe molecules, many biochips include a 2-dimensional array of 3-dimensional polymeric anchoring structures (for example, polyacrylamide gel pads) attached to the surface of the substrate. Probe molecules such as oligonucleotides covalently attach to polyacrylamide-anchoring structures by forming an amide, ester or disulfide bond between the biomolecule and a derivatized polymer comprising the cognate chemical group. Covalent attachment of probe molecules to the polymeric anchoring structure is usually performed after polymerization and chemical cross-linking of the polymer to the substrate is completed.
Existing apparatus for performing thermally-controlled biological reactions on a substrate surface are deficient in that they either require unacceptably large volumes of sample fluid to operate properly, cannot accommodate substrates as large as or larger than a conventional microscope slide, cannot independently accommodate a plurality of independent reactions, or cannot accommodate a substrate containing hydrogel-based microarrays. Most existing apparatus also do not allow introduction of fluids in addition to the sample fluid such as wash buffers, fluorescent dyes, etc., into the reaction chamber. Disposable apparatus require disassembly and reapplication of a new apparatus to the substrate surface every time a new fluid must be introduced. Other existing apparatus are difficult to use in a laboratory environment because they cannot be loaded with standard pipet tips and associated pipettor apparatus.
Many existing apparatus also exhibit unacceptable reaction reproducibility, efficiency, and duration. Reaction reproducibility may be adversely affected by bubble formation in the reaction chamber or by the use of biologically incompatible materials for the reaction chamber. Reaction duration and efficiency may be adversely affected by the presence of concentration gradients in the reaction chamber.
Bubbles can form on introduction of sample fluid to the reaction chamber, at elevated temperatures during the reaction due to the potential high gas content of the fluid, or by outgassing of the reaction chamber materials. When gas bubbles extend over the substrate surface in an area containing biologically reactive sites, the intended reaction may intermittently fail or yield erroneous results because the intended concentration of the sample fluid mixture has been compromised by the presence of gas bubbles. To aggravate the problem, gas bubbles in the reaction chamber attempt to expand at elevated temperatures during the reaction and periodically cause the seal between the substrate surface and reaction chamber apparatus to fail, allowing leakage and evaporation of the sample fluid.
Biologically incompatible reaction chamber materials may cause unacceptable reaction reproducibility, by interacting with the sample fluid, thus causing the intended reaction to intermittently fail or yield erroneous results.
Incomplete mixing of the sample fluid can introduce concentration gradients within the sample fluid that adversely impact reaction efficiency and duration. This effect is most pronounced when there is depletion of target molecules in the local volume surrounding a biologically reactive site. During a biological reaction, the probability that a particular target molecule will bind to a complementary (immobilized) probe molecule is determined by the given concentration of target molecules present within the sample fluid volume, the diffusion rate of the target molecule through the reaction chamber, and the statistics of interaction between the target molecule and the complementary probe molecule. For diagnostic assays, target DNA molecules are often obtained in minute ( less than picomol) quantities. In practice, it can take tens of hours for a hybridization reaction to be substantially complete at the low target nucleic acid molecule levels available for biological samples. Concentration gradients further exacerbate this problem.
U.S. Pat. No. 5,948,673 to Cottingham discloses a self-contained multi-chamber reactor for performing both DNA amplification and DNA probe assay in a sealed unit wherein some reactants are provided by coating the walls of the chambers and other reactants are introduced into the chambers prior to starting the reaction in order to eliminate flow into and out of the chamber. Unfortunately, no provisions are made for pressurization or mixing of the sample fluid introduced to the chambers, and the apparatus cannot accommodate substrates including microscope slides.
There remains a need in the art for methods and apparatus for performing biological reactions on a substrate surface that use a low volume of sample fluid, accommodate substrates as large as or larger than a conventional microscope slide, accommodate a plurality of independent reactions, and accommodate a substrate surface having one or more hydrogel-based microarrays attached thereto. There also remains a need in the art for an apparatus that allows introduction of fluids in addition to sample fluid into each reaction chamber via standard pipet tips and associated pipettor apparatus. There also remains a need in the art for such an apparatus that increases reaction reproducibility, increases reaction efficiency, and reduces reaction duration. These needs are particularly striking in view of the tremendous interest in biochip technology, the investment and substantial financial rewards generated by research into biochip technology, and the variety of products generated by such research.
The present invention provides methods and apparatus for performing biological reactions on a substrate surface that use a low volume of sample fluid, accommodate substrates as large as or larger than a conventional microscope slide, accommodate a plurality of independent reactions, and accommodate a substrate surface having one or more hydrogel-based microarrays attached thereto. The invention further provides an apparatus that allows introduction of fluids in addition to sample fluid into each reaction chamber via standard pipet tips and associated pipettor apparatus. The invention further provides an apparatus that increases reaction reproducibility, increases reaction efficiency, and reduces reaction duration.
The invention broadly comprises a base plate having a first surface and a cavity disposed in the first surface, wherein the cavity comprises one or more well structures and a biochip comprising one or more microarrays of biologically reactive sites disposed on a first surface can be inserted into the apparatus such that the first surface of the biochip is in direct communication with the well structures and is removably clamped to the base plate using a compression plate. A sealing member is disposed between the first surface of the substrate and the first surface of the base plate in each well structure, thereby defining one or more reaction chambers. Each well structure has at least two fluid ports for introducing fluid samples into and removing, fluid samples from the reaction chambers. The invention further comprises a seal for the fluid ports.
A preferred embodiment of the invention is configured to accommodate a biochip comprising a standard microscope slide having a plurality of hydrogel-based microarrays attached thereto. A further preferred embodiment of the apparatus includes the biochip.
In preferred embodiments of the present invention, the sealing member around the perimeter of each well structure comprises an O-ring or sheet of gasket material.
In further preferred embodiments, the fluid ports allow introduction of fluid sample via a standard pipet tip or tubing. In still further preferred embodiments, the fluid ports allow interface to an external pumping system that provides mixing and pressurization of the fluid in each reaction chamber to provide uniform target molecule concentration and dissolve gas bubbles, respectively.
In preferred embodiments, the fluid port seal comprises a layer of flexible, thermally conductive material on which is disposed a layer of pressure-sensitive adhesive.
In other preferred embodiments of the invention, the biological compatibility of the base plate material is enhanced by the addition of a biologically compatible surface coating to the first surface of the base plate. The adhesion of the surface coating to the first surface of the base plate may be further enhanced by application of a layer of primer on the first surface of the base plate prior to application of the surface coating.
In further preferred embodiments of the invention, the compression plate is removably affixed to the base plate by a plurality retaining pins disposed along the perimeter of the base plate which fit into corresponding locking apertures disposed along the perimeter of the retaining plate. In yet further preferred embodiments, the compression plate comprises a cavity wherein a compliance layer is seated.
In preferred embodiments of the microfluidic reaction apparatus, the retaining plate, compression plate and compliance layer further comprise one or more viewing ports corresponding in position to the reaction chambers for observation of the biological reactions taking place inside the reaction chambers.
The invention is advantageously used for performing thermally controlled biological reactions, and in preferred embodiments comprises a heating element and a thermal cycling device.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.